title: High-level gene transfer to cord blood progenitors using gibbon ape leukemia virus pseudotype retroviral vectors and an improved clinically applicable protocol.
abstract: The best methods for transducing hematopoietic progenitor cells usually involve either direct co-cultivation with virus-producing cells or human stromal supportive cells. However, these methods cannot be safely or easily applied to clinical use. Therefore, we aimed at improving retrovirus-mediated gene transfer into hematopoietic progenitors derived from cord blood CD34+ cells using viral supernatant to levels achieved at least with direct co-cultivation and under conditions that are suitable for clinical applications. In a first set of experiments, CD34+ cells were infected with supernatant containing amphotropic retroviral particles carrying the nls-lacZ reporter gene and the effects of centrifugation, cell adhesion to fibronectin, and Polybrene on the transduction of both clonogenic progenitors (CFC) and long-term culture initiating cells (LTC-IC) were studied. Transduction efficiency was evaluated on the percentage and total number of progenitors expressing the beta-galactosidase activity. Results show that a 48-hr infection of CD34+ cells with viral supernatant combining centrifugation at 1000 x g for 3 hr followed by adhesion to fibronectin allows transduction levels for both CFC and LTC-IC to be reached that are as good as using direct co-cultivation. In a second set of experiments, CD34+ cells were infected using this optimized protocol with pseudotyped retroviral particles carrying the gibbon ape leukemia virus (GALV) envelope protein. Under these conditions, between 50 and 100% of CFC and LTC-IC were transduced. Thus, we have developed a protocol capable of highly transducing cord blood progenitors under conditions suitable for a therapeutical use.
authors.affiliation: Biologie et Thérapie des Pathologies Immunitaires, ERS CNRS 107 C.E.R.V.I., CHU Pitié Salpétrière, Paris, France.
authors.forename: M
authors.initials: M
authors.lastname: Movassagh
authors.forename: C
authors.initials: C
authors.lastname: Desmyter
authors.forename: C
authors.initials: C
authors.lastname: Baillou
authors.forename: S
authors.initials: S
authors.lastname: Chapel-Fernandes
authors.forename: M
authors.initials: M
authors.lastname: Guigon
authors.forename: D
authors.initials: D
authors.lastname: Klatzmann
authors.forename: F M
authors.initials: FM
authors.lastname: Lemoine
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D002460:Cell Line; D002498:Centrifugation; D018920:Coculture Techniques; D003470:Culture Media; D005312:Fetal Blood / Q000166:cytology / Q000187:drug effects / Q000821:virology*; D005353:Fibronectins / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000627:therapeutic use*; D006583:Hexadimethrine Bromide / Q000494:pharmacology; D006801:Humans; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics* / Q000254:growth & development; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000821:virology*
chemical_list: D018952:Antigens, CD34; D003470:Culture Media; D005353:Fibronectins; D006583:Hexadimethrine Bromide